The complex diterpenoid Taxol™ (Bristol-Myers Squibb; common name paclitaxel) (Wani et al., J. Am. Chem. Soc. 93:2325-2327, 1971) is a potent antimitotic agent with excellent activity against a wide range of cancers, including ovarian and breast cancer (Arbuck and Blaylock, Taxol: Science and Applications, CRC Press, Boca Raton, 397-415, 1995; Holmes et al., ACS Symposium Series 583:31-57, 1995). Paclitaxel was isolated originally from the bark of the Pacific yew (Taxus brevifolia). For a number of years, paclitaxel was obtained exclusively from yew bark, but low yields of this compound from the natural source coupled to the destructive nature of the harvest, prompted new methods of paclitaxel production to be developed.
Total chemical syntheses of paclitaxel have been achieved (for review, see, Kingston et al., Prog. Chem. Org. Nat. Prod. 84:56-225, 2002) but the yields of the drug by this method are too low to be practical. Paclitaxel currently is produced primarily by chemical semisynthesis from advanced taxane metabolites (Holton et al., Taxol: Science and Applications, CRC Press, Boca Raton, 97-121, 1995; Hezari and Croteau, Planta Medica, 63:291-295, 1997) that are isolated from the needles (a renewable resource) of various Taxus speciea. However, at least because of the increasing demand for this drug both for use earlier in the course of cancer intervention and for new therapeutic applications (Goldspiel, Pharmacotherapy 17:110S-125S, 1997), high-yield, cost-effective methods of paclitaxel production continue to be needed. Some have proposed isolating paclitaxel from alternative biological sources, such as the endophytic fungi, Taxomyces andreanae (Stierle et al., J. Nat. Prod 58:1315-1324, 1995), or from Taxus cell cultures (Ketchum et al., Biotechnol. Bioeng. 62:97-105, 1999). However, these methods are also too inefficient to produce sufficient quantities of the drug and have had limited commercial success.
Improving the production yield of paclitaxel from any biological system, whether intact organisms (such as, Taxus plants or paclitaxel-producing fungi) or cell cultures, would be facilitated by a detailed understanding of the paclitaxel biosynthetic pathway. The paclitaxel biosynthetic pathway is complex and believed to involve nearly 20 distinct steps (Floss and Mocek, Taxol: Science and Applications, CRC Press, Boca Raton, 191-208, 1995; and Croteau et al., Curr. Top. Plant Physiol. 15:94-104, 1996). However, relatively few of the enzymatic reactions and intermediates of this complicated pathway have been defined in detail.
The first committed enzyme of the paclitaxel pathway is believed to be taxadiene synthase (Koepp et al., J. Biol. Chem. 270:8686-8690, 1995), which cyclizes the common precursor geranylgeranyl diphosphate (Hefner et al., Arch. Biochem. Biophys. 360:62-74, 1998) to taxadiene (FIG. 1). The cyclized intermediate (i.e., taxa-4(5),11(12)-diene) subsequently undergoes modification involving at least eight oxygenation steps, a formal dehydrogenation, an epoxide rearrangement to an oxetane, and several acylations (Floss and Mocek, Taxol: Science and Applications, CRC Press, Boca Raton, 191-208, 1995; and Croteau et al., Curr. Top. Plant Physiol. 15:94-104, 1996). Taxadiene synthase has been isolated from T. brevifolia and characterized (Hezari et al., Arch. Biochem. Biophys. 322:437444, 1995), the mechanism of action defined (Lin et al., Biochemistry 35:2968-2977, 1996), and the corresponding cDNA clone isolated and expressed (Wildung and Croteau, J. Biol. Chem. 271:9201-9204, 1996).
The second specific step of paclitaxel biosynthesis is believed to be an oxygenation (hydroxylation) reaction that introduces a hydroxyl group to position 5 of taxa-4(5),11(12)-diene to produce taxa-4(20),11(12)-dien-5α-ol. Using a crude Taxus microsome preparation, Hefner et al. (Methods Enzymol. 272:243-250, 1996) demonstrated a microsomal activity that catalyzed the stereospecific hydroxylation of taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5α-ol (with double-bond rearrangement) (Hefner et al., Chem. Biol., 3:479-489, 1996). This microsomal activity was attributed to one or more cytochrome P450 oxygenases (Hefner et al., Chemistry and Biology 3:479-489, 1996). Cytochrome P450 oxygenases are enzymes that have a unique sulfur atom ligated to the heme iron and that, when reduced, form carbon monoxide (CO) complexes. When complexed to carbon monoxide, cytochrome P450 proteins display a major absorption peak (Soret band) near 450 nm.
Taxus microsomal preparations were further shown to catalyze the hydroxylation of taxadiene or taxadien-5α-ol to the level of a pentaol (Hefner et al., Methods Enzymol. 272:243-250, 1996; Lovy Wheeler et al., Arch. Biochem. Biophys., 390:265-278, 2001). These results suggested that the paclitaxel biosynthetic pathway included at least five distinct cytochrome P450 taxoid oxygenases in the early parts of the pathway (Hezari et al., Planta Med. 63:291-295, 1997). Later steps of the paclitaxel biosynthetic pathway are thought to include at least three additional oxygenation steps (C1 and C7 hydroxylations and an epoxidation at C4-C20). These steps also are believed to be catalyzed by cytochrome P450 enzymes, but these reactions reside too far down the pathway to observe in microsomes by current experimental methods (Croteau et al., Curr. Topics Plant Physiol. 15:94-104, 1995; Hezari et al., Planta Med. 63:291-295, 1997 Lovy Wheeler et al., Arch. Biochem. Biophys., 390:265-278, 2001). Since Taxus (yew) plants and cells do not appear to accumulate taxoid metabolites bearing fewer than six oxygen atoms (e.g., hexaol or epoxypentaol) (Kingston et al., Prog. Chem. Org. Nat. Prod. 61:1-206, 1993), such intermediates must be rapidly transformed down the pathway, indicating that the oxygenations (hydroxylations) are relatively slow pathway steps.
Taxus microsome preparations contain hundreds of different proteins, including an estimated 30 to 50 similar cytochrome P450 oxygenases (Hefner et al., Methods Enzymol. 272:243-250, 1996). Biochemical purification of cytochrome P450 enzymes from Taxus microsomes (Hefner et al., Methods Enzymol. 272:243-250, 1996) is not practical, at least, because the numerous P450 cytochrome oxygenases present in this cell fraction have very similar physical properties (Mihaliak et al., Methods Plant Biochem. 9:261-279, 1993). With no useful biochemical means to distinguish among the many microsomal P450 oxygenases, it is not feasible to sufficiently purify any one enzyme to obtain even short peptide sequences. As a result, other methods are needed to isolate and characterize these important enzymes at the molecular level.
Differential display reverse transcription PCR (DD-RT PCR) has been used to isolate methyl jasmonate-induced nucleic acids encoding taxoid oxygenases of the paclitaxel biosynthetic pathway (see, for example, PCT Pub. No. WO01/34780). Several of the encoded oxygenase enzymes have been expressed and functionally characterized (PCT Pub. No. WO01/34780; Schoendorf et al., Proc. Natl. Acad. Sci. USA, 98:1501-1506, 2001; Jennewein et al., Proc. Natl. Acad. Sci. USA, 98:13595-13600, 2001; Jennewein et al., Arch Biochem. Biophys., 413:262-270, 2003). However, transcripts encoding taxoid oxygenases that are not, or only weakly, induced are likely to be missed by the DD-RT PCR technique.
Paclitaxel is an important drug that is not efficiently produced using current methods. Genetic engineering and recombinant technologies offer ways to increase paclitaxel and taxoid yields. To capitalize on these technologies, there is a continuing need to identify and isolate the genes encoding the enzymes of the paclitaxel biosynthetic pathway, including, for example, the numerous oxygenase enzymes, and for methods of using such genes and enzymes to produce paclitaxel and its intermediates.